When the product is prepared by cell cultivation during bioprocess, the growth and the cellmorphology which are customarily sampled from the tank and then observed by an off-line microscope, are very important monitoring parameters. Recently, quantified information on the growth and the morphology may be obtained with the development of electronic technique, which enables the conversion of images into digital signals to be processed by a computer. However, the foregoing methods merely make use of off-line sampling and therefore no dynamic observation can be made. Particularly, in the case of large scale animal cells cultivation, wherein prevention of foreign microbes from contamination is the primary concern in the process, therefore, frequent sampling should be avoided. In such a case, it will result in poor dynamic observation and difficult process analysis.
Thus, there is an urgent need in the art for a biochemical reactor, which is equipped with an in-situ cell examination microscope with excellent dynamic observation, high resolution and easy process analysis. An in-situ cell examination microscope which is inserted into a fermenter should meet the following requirements: 1) the resolution and visual field should be suitable for distinguishing bacteria, fungi and animal cells, dead cells and living cells, and conducting a morphological analysis correspondingly; 2) there should be a maximum concentration limit due to variation of cell concentration during the course of cell culture; 3) the problem that microorganisms grow or adhere to the surface of the lens after the probe of the microscope has been inserted into a fermenter for a long time should be overcome; 4) the structure of the microscope should withstand sterilization and sterile operations of the biochemical reactor; 5) the microscope should be able to convert and process real-time digital image signals in real time according to the requirement of the reactor.
An in-situ microscopic device based on fluorescence-excited process is disclosed by Dr Christoph Bittner from Germany. The main working principle of the device is shown in FIG. 1, wherein the monochromatic UV light emitted by a laser is filtered by a lens L1 and a pinhole space, and then passes through two lenses, L2 and L3 before it is reflected by a bichromatic glass onto an object lens which directs the light onto the surfaces of the cells in a biochemical reactor chamber. The fluorescence generated by cells due to fluorescence effect converges at the object lens, passes the bichromatic glass, a reflector and a wave filter in sequence, and finally develops an image in a synchronization camera. Since the cells in the biochemical reactor are rotating at high speed, it is necessary for the synchronization camera to have a very short exposure time, usually less than 2 ms (depending on the magnifying power). The advantage of this process is omission of any complicated mechanical sampling device. However, contrast and resolution for the images of transparent cells are poor due to the bright-field illumination used in this process. Furthermore, since the manner of single-wavelength fluorescence-excitation generated through the excitation of a beam for high-speed photo-shooting is employed, it will cost high to change the stimulating beam wavelength into a different one.
A microscope based on in-situ sampling is disclosed in U.S. Pat. No. 6,809,862 in 2004. As shown in FIG. 2, a sampling window 4 keeps moving to and fro. When the sampling window 4 moves to the place where a microscope 16 and a light source are located, the cell culture in a biochemical reactor 10 will flow into an examination chamber 12. When the sampling window 4 moves to another place, the cell culture in the examination chamber 12 is relatively stable, for micro-examination. To replace the microlens conveniently, a rinsing chamber is designed as shown in FIG. 3. The microlens and the sampling window 4 can be drawn into the rinsing chamber by pulling a rod 2. There are holes 8 in the wall of the rinsing chamber for introducing steam or disinfectant for sterilization or rinsing. The microlens also can be replaced conveniently in-situ. Likewise, contrast and resolution for the images of transparent cells are poor due to the bright-field illumination used in this process. Furthermore, since the illumination system is positioned within the biochemical reactor, it is not convenient to replace the light source and change the wavelength of the excitation light, and unable to distinguish different species of cells, for example, dead cells and living cells. Additionally, frequent sampling and sterilization may contaminate the biological culture process.
An in-situ cell examination microscope is disclosed in DE 10350243 in 2004, which makes use of SLD (Super Light Diode) illumination, multimode fiber transmission and high-speed synchronization shooting. A light beam emitted by a laser or a light emitting diode is transmitted by a multimode fiber into a biochemical reactor to illuminate the cells in the biochemical reactor (the reactor), and the light reflected by the cells converges at an object lens and then develops into an image in a CCD camera. Since the cells continuously move in the biochemical reactor, the exposure time of the camera has to be short enough to achieve an instant capture of a cell image. The images captured by the camera are sent to a computer for processing. Likewise, contrast and resolution for the images of transparent cells are poor due to the bright-field illumination used in this process. High-speed agitation in the biochemical reactor entails the use of a high-speed camera. There is no sampling device herein which may restrict the field depth, the effect of which thus can not be eliminated, just as in the case shown in FIG. 1. Additionally, since a complex SLD (Super Light Diode) is used as the light source, the driving and controlling device of the light source is complicated, resulting in high cost and difficulty in replacing the light source and changing the excitation wavelength.
Apparently, there is an urgent need in the art for a biochemical reactor equipped with an in-situ cell examination microscope with excellent dynamic examination, high resolution and easy process analysis.
On the other hand, it is important to monitor the biomass in the production of living cells. Especially, the advancement in metabolic engineering research has been successfully applied in various fields including microbial breeding, optimization of existing processes, development of new products and environment management. The new trend is to combine the study on fermentation with that on the mechanism of intracellular processes, and to combine sensing technology, computer technology, isotope technology with various detection technologies used in laboratory, so as to make real-time metabolic flux analysis of the processes. However, due to the use of composite culture medium containing solid particles in fermentation and the difficulty in distinguishing dead cells from living ones, the amount of living cells, which is significant for determining a real-time metabolic flow, has to be measured with a manual means used in a laboratory or with an indirect means suffering from a large error. It is urgent to develop a new technical route by combining the in-situ technology for measuring living cell concentration with metabolic flux analysis.
Thus, there is a lack of a biochemical reactor in the art, which is adapted for measuring the amount of living cells therein for correlation analysis.
In addition, a fermentation process is customarily optimized and scaled up on the basis of a static concept wherein extracellular parameters are measured and used to choose the optimal point for process control. This is essentially no more than an extension of the macro-dynamic concept in the field of chemical engineering to the field of fermentation engineering. Along with the intensive development of research on life science, mechanisms of intracellular processes are getting more and more clear. However, the research focused merely on physiological regulation mechanism can usually reveal just a local aspect or a temporary feature of physiological regulation. Highly branched and dispersed research can not play a decisive role in controlling, optimizing and scaling up the whole process of a biochemical reactor. In an attempt to solve the above systematic problems, after studying the characteristics of cellular process in a biochemical reactor, the present inventors have devoted to the study on the multi-scale problem of cellular process, and have provided an optimizing technique based on correlated parameters of a fermentation process at multi-levels and a scaling-up technique based on the adjustment of multiple parameters of a fermentation process.
Along with the intensive development of the foregoing study on basic theories, there is an urgent need for sensing technology and computer technology to follow the development of a process, and to design a new-concept fermenter for the multi-scale study of a biological process. The fermenter is capable of in-situ detecting or controlling multiple parameters based on the monitoring of the material stream in a biochemical reactor. Furthermore, a software package is developed, which is suitable for various reactors, combines various process theories and controlling theories, and could be used for process analysis and optimizing operations of a fermentation process.
On the other hand, there is a lack of a novel biochemical reactor in the art, which is adapted for multi-parameter correlation analysis.
In summary, there is an urgent need in the art for a biochemical reactor equipped with an in-situ cell examination microscope with excellent dynamic examination, high resolution and easy process analysis.